P1206: A SINGLE-CELL MULTI-OMIC AND SPATIAL ATLAS OF NODAL B CELL NON-HODGKIN LYMPHOMAS REVEALS PLASTICITY IN B CELL MATURATION AS A DRIVER OF INTRATUMOR HETEROGENEITY
Authors: Donnacha Fitzgerald, Artur Kibler, Marc-A. Baertsch, Tobias Roider, Harald Vohringer, Anastasiia Horlova, Anna Mathioudaki, Bettina Budeus, Mareike Knoll, Erin Chung, Johannes Mammen, Leandra Caille, Verena Passerini, Felix Czernilofsky, Linsha LI, Oliver Weigert, Judith B. Zaugg, Gary P. Nolan, Marc Seifert, Wolfgang Huber, Sacha Dietrich
Journal: HemaSphere (2023)

Topic: 20. Lymphoma Biology & Translational Research Background: Intratumor heterogeneity (ITH) is increasingly recognized as an essential factor in the variability of B cell non-Hodgkin lymphoma (B-NHL) patient outcomes. It is well established that distinct nodal B cell non-Hodgkin lymphoma (NBNHL) entities originate from different stages of the humoral affinity maturation process, but the drivers of ITH are not well understood. Aims: In this study, we aimed to characterize ITH in B-NHL and investigate its relationship with the nodal B cell affinity maturation process. Methods: 51 diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and non-malignant reactive lymph node (rLN) samples from distinct patients were sequenced with CITE-Seq (coupling single-cell RNA-seq with surface epitope profiling). Transcription factor activities and copy number variants (validated against targeted DNA sequencing) were inferred computationally from gene expression, while B cell receptor clones were determined with VDJ profiling. Spatial relationships were determined with highly multiplexed immunochemistry (CODEX). Informed by gene expression signatures from RNA sequencing of FACS-identified nodal B cell maturation states, a CITE-Seq reference map was constructed in reactive lymph nodes. This reference map was used to classify maturation states in the malignant samples with logistic regression, which were verified and refined by their maturation marker profiles. Using the shared features between CITE-Seq and CODEX panels, maturation states were similarly classified in the spatial data and their microenvironments were characterized with cellular neighborhood analysis. (Fig A) Results: As in the non-malignant context, variability in maturation states was also observed among malignant cells in most NBNHL tumors. BCR-sequencing and light chain restriction confirmed that different intra-tumor maturation states observed in samples were monoclonal, and therefore arose from the same cell of origin. Although commonalities in composition existed between samples within entities, such as an abundance of germinal center (GC) states in DLBCL and memory states in MZL, we observed high variability in maturation state proportions between samples. For example, ABC DLBCL and FL tumors frequently contained both GC and post-GC subpopulations. Transcription factor mediators of the B cell maturation process remained active in malignancy. Copy number variation was observed between maturation states of the same tumor, indicating genetic variants can arise at sequential stages of the B cell maturation process in existent malignancy. Intratumor maturation states typically occupied distinct spatial niches. Although nodal architecture disruption and microenvironmental variation were observed across tumors, tumor cells often maintained proximity with follicular dendritic and follicular helper T cells, key mediators of the affinity maturation process. Summary/Conclusion: Our findings reveal that B cell maturation is not halted during malignancy, but remains a continuous and divergent process central to inter- and intratumor variation. Intratumor maturation states can be subject to genetic variation and occupy distinct spatial niches while retaining the cellular interactions and signals which enable the maturation process. (Fig B)Keywords: B cell development, B cell lymphoma, B cell subsets, OMICS